Screening of biogenic amine production by lactic acid bacteria isolated from grape must and wine

The potential to produce the biogenic amines tyramine, histamine and putrescine, was investigated for lactic acid bacteria (LAB) of various origin, including commercial malolactic starter cultures, type strains and 78 strains isolated from Spanish grape must and wine. The presence of biogenic amines in a decarboxylase synthetic broth was determined by reverse-phase high performance liquid chromatography (RP-HPLC). Tyramine was the main amine formed by the LAB strains investigated. Leuconostoc strains were the most intensive tyramine formers. No potential to form biogenic amines was observed in Oenococcus oeni strains. Two strains of Latobacillus buchneri were associated with putrescine formation. None of the lactic acid bacteria produced histamine. According to these in vitro results, the commercial starter bacteria analyzed did not produce histamine, tyramine and putrescine.     

发酵剂抑制发酵肉制品中酪胺形成机制及效果的研究进展

酪胺是以酪氨酸为前体物质，经脱羧后形成的一种生物胺。消费者从食品中摄入过量的酪胺会导致头痛、高血压等健康问题。发酵肉制品由于富含蛋白质，且经长期发酵产生了复杂的微生物菌群和大量的酪氨酸，因此易形成酪胺并在产品中大量积累。应用不产生酪胺或具有降解酪胺功能的发酵剂是降低发酵肉制品中酪胺含量的新方法。本文介绍了发酵肉制品中酪胺的形成及其影响因素，重点综述了发酵剂的种类、特性和发酵剂抑制酪胺形成的机制和效果，旨在为通过发酵剂法抑制发酵肉制品中的酪胺形成与积累、提高发酵食品安全提供理论支持。     

Sequencing of the tyrosine decarboxylase cluster of Lactococcus lactis IPLA 655 and the development of a PCR method for detecting tyrosine decarboxylating lactic acid bacteria

The enzymatic decarboxylation of tyrosine produces tyramine, the most abundant biogenic amine in dairy products-especially in cheeses. The screening of lactic acid bacteria isolated from different artisanal cheeses and a number of microbial collections identified 22 tyramine-producing strains belonging to different genera. The Lactococcus lactis strain IPLA 655 was selected, and the genes encoding a putative tyrosyl tRNA synthetase, a tyrosine decarboxylase (tdcA), and a tyrosine-tyramine antiporter, found together as a cluster, were sequenced. The disruption of tdcA yielded a strain unable to produce tyramine. Comparison of the L. lactis IPLA 655 tdcA gene with database tdcA sequences led to the design of two primers for use in a PCR method that identified potential tyramine-producing strains. The proposed method can use purified DNA, isolated colonies, milk, curd, and even cheese as a template. Molecular tools for the rapid detection of tyramine-producing bacteria at any time during the fermentation process could help prevent tyramine accumulation in fermented foods. The proposed technique could be of great use to the food industry.     

Updated molecular knowledge about histamine biosynthesis by bacteria

Histamine poisoning is caused by the ingestion of food containing high levels of histamine, a biogenic amine. Histamine could be expected in virtually all foods that contain proteins or free histidine and that are subject to conditions enabling microbial activity. In most histamine-containing foods the majority of the histamine is generated by decarboxylation of the histidine through histidine decarboxylase enzymes derived from the bacteria present in food. Bacterial histidine decarboxylases have been extensively studied and characterized in different organisms and two different enzymes groups have been distinguished, pyridoxal phosphate- and the pyruvoyl-dependent. Pyridoxal phosphate-dependent histidine decarboxylases are encountered in gram-negative bacteria belonging to various species. Pyruvoyl-dependent histidine decarboxylases are found in gram-positive bacteria and specially in lactic acid bacteria implicated in food fermentation or spoilage. The molecular organization of the genes involved in histamine production have been elucidated in several histamine-producer bacteria. This molecular knowledge has led to the development of molecular methods for the rapid detection of bacteria possessing the ability to produce histamine. The detection of histamine-producer bacteria is of great importance for its potential health hazard as well as from an economic point of view since products exceeding recommended limits can be refused in commercial transactions.     

环境因素对屎肠球菌产酪胺的影响

酪胺是发酵食品中广泛存在的、毒性较强的一种生物胺。屎肠球菌作为发酵剂或污染菌广泛存在于发酵食品中,大多数具有产生酪胺的能力,成为发酵食品的潜在安全隐患。该研究以一株黄酒酒曲中分离的产酪胺屎肠球菌为对象,选取葡萄糖、酪氨酸、温度、pH值和酒精含量5个因素,研究这些因素对该菌生长和酪胺合成的影响。结果表明,葡萄糖或酪氨酸添加量对菌体的生长影响不大,温度、环境pH值和酒精含量对菌体生长的影响较显著。酪氨酸对菌体产酪胺促进作用明显,温度＜20 ℃,环境pH值＞5,酒精含量＞10%vol时都会减少酪胺的产生。     

Improved screening procedure for biogenic amine production by lactic acid bacteria

An improved screening plate method for the detection of amino acid decarboxylase-positive microorganisms (especially lactic acid bacteria) was developed. The suitability and detection level of the designed medium were quantitatively evaluated by confirmation of amine-forming capacity using an HPLC procedure. The potential to produce the biogenic amines (BA) tyramine, histamine, putrescine, and cadaverine, was investigated in a wide number of lactic acid bacteria (LAB) of different origin, including starter cultures, protective cultures, type strains and strains isolated from different food products. Also, several strains of Enterobacteriaceae were examined. Modifications to previously described methods included lowering glucose and sodium chloride concentrations, and increasing the buffer effect with calcium carbonate and potassium phosphate. In addition, pyridoxal-5-phosphate was included as a codecarboxylase factor for its enhancing effect on the amino acid decarboxylase activity. The screening plate method showed a good correlation with the chemical analysis and due to its simplicity it is presented as a suitable and sensitive method to investigate the capacity of biogenic amine production by LAB. Tyramine was the main amine formed by the LAB strains investigated. Enterococci, carnobacteria and some strains of lactobacilli, particularly of Lb. curvatus. Lb. brevis and Lb. buchneri, were the most intensive tyramine formers. Several strains of lactobacilli, Leuconostoc spp., Weissella spp. and pediococci did not show any potential to produce amines. Enterobacteriaceae were associated with cadaverine and putrescine formation. No significant histamine production could be detected for any of the strains tested.     

Contaminant lactic acid bacteria of dry sausages produce histamine and tyramine

Exogenous biogenic amines present a food poisoning hazard in fermented foods especially with additional risk factors, e.g. amine oxidase inhibiting drugs, alcohol and gastrointestinal diseases. Forty-two lactic acid bacteria (LAB) strains were isolated from seven dry sausages during ripening (0 day, 21 day and 49 day). Their ability to produce histamine and tyramine was studied by HPLC detection from broth cultures after 2 days incubation.

The tyramine and histamine concentrations in sausages increased during fermentation. 10 of 42 LAB strains produced 402–1087 ppm tyramine. Four of these 10 contaminant LABs were also histamine-positive (725–1083 ppm). Most of the amine positive strains were found in sausages at the end of ripening and with highest amine levels.

On the basis of these results the contaminant LABs play an important role in tyramine and histamine formation during the ripening of dry sausages. Therefore it could be possible to decrease the levels of amines formed by limiting the initial level and growth of these contaminant bacteria.     

EVALUATION OF BIOGENIC AMINES FORMATION BY PROTEOLYTIC ENTEROCOCCI STRAINS ISOLATED FROM RAW DROMEDARY MILKS FROM SOUTHERN ALGERIA

Twenty-three strains of lactic acid enterococci isolated from dromedary raw milks in Southern Algeria were investigated for their proteolytic activity and ability to produce biogenic amines by decarboxylating amino acids: histidine, tyrosine and ornithine. All strains tested displayed proteolytic activities, but none of them were able to produce detectable biogenic amines in the usual adequate laboratory media, namely, Joosten and Northolt medium and Leuschner medium. Polymerase chain reaction experiments indicate a positive correlation between the absence of the hdc and odc genes and the lack of histamine and putrescine production for all tested strains. By contrast, all strains appeared to possess tdc gene, but no correlation was found with a negative expression of tyrosine decarboxylase.

PRACTICAL APPLICATIONS

In foods, biogenic amines are mainly generated by decarboxylation of the corresponding amino acids through substrate-specific enzymes of the microorganisms present in this environment, particularly histidine, tyrosine, lysine, ornithine and arginine, and the capability of enterococci and some other lactic acid bacteria to produce biogenic amines has been described. For this purpose, several qualitative and quantitative methods to determine biogenic amines production by these microorganisms have been developed. There is great interest to identify and to characterize the bacteria strains intended for an industrial use and to verify if they are potentially able to produce biogenic amines in order to prevent it's synthesis and limit the poisoning risk.     

Lactobacillus casei strains isolated from cheese reduce biogenic amine accumulation in an experimental model

Tyramine and histamine are the biogenic amines (BAs) most commonly found in cheese, in which they appear as a result of the microbial enzymatic decarboxylation of tyrosine and histidine respectively. Given their toxic effects, their presence in high concentrations in foods should be avoided. In this work, samples of three cheeses (Zamorano, Cabrales and Emmental) with long ripening periods, and that often have high BA concentrations, were screened for the presence of BA-degrading lactic acid bacteria (LAB). Seventeen isolates were found that were able to degrade tyramine and histamine in broth culture. All 17 isolates were identified by 16S rRNA sequencing as belonging to Lactobacillus casei. They were typed by plasmid S1-PFGE and genomic macrorestriction-PFGE analysis. Two strains (L. casei 4a and 5b) associated with high degradation rates for both BAs were selected to test how this ability might affect histamine and tyramine accumulation in a Cabrales-like mini-cheese manufacturing model. The quantification of BAs and the monitoring of the strains' growth over ripening were undertaken by RP-HPLC and qPCR respectively. Both strains were found to reduce histamine and tyramine accumulation. These two strains might be suitable for use as adjunct cultures for reducing the presence of BAs in cheese.     

Phenotypic and genetic evaluations of biogenic amine production by lactic acid bacteria isolated from fish and fish products

In this work, biogenic amine production (histamine, tyramine and putrescine) by a collection of 74 lactic acid bacteria of aquatic origin has been investigated by means of amino acid decarboxylation by growth on decarboxylase differential medium, biogenic amine detection by thin-layer chromatography (TLC) and decarboxylase gene detection by PCR. None of the evaluated strains showed neither production of histamine and putrescine, nor presence of the genetic determinants encoding the corresponding decarboxylase activities. However, the tyrosine decarboxylase gene (tdc) was present in all the enterococcal strains, and tyramine production was detected by TLC in all of them but Enterococcus faecium BCS59 and MV5. Analysis of the tyrosine decarboxylase operon of these strains revealed the presence of an insertion sequence upstream tdc that could be responsible for their lack of tyrosine decarboxylase activity.     

Production of biogenic amines by lactic acid bacteria: screening by PCR, thin-layer chromatography, and high-performance liquid chromatography of strains isolated from wine and must

Biogenic amines are frequently found in wine and other fermented food. We investigated the ability of 133 strains of lactic acid bacteria isolated from musts and wines of different origins to produce histamine, tyramine, and putrescine. We detected the genes responsible for encoding the corresponding amino acid decarboxylases through PCR assays using two primer sets for every gene: histidine decarboxylase (hdc), tyrosine decarboxylase (tdc), and ornithine decarboxylase (odc); these primers were taken from the literature or designed by us. Only one strain of Lactobacillus hilgardii was shown to possess the hdc gene, whereas four strains of Lactobacillus brevis had the tdc gene. None of the Oenococcus oeni strains, the main agents of malolactic fermentation, was a biogenic amine producer. All PCR amplicon band-positive results were confirmed by thin-layer chromatography and high-performance liquid chromatography analyses.     

Molecular methods for the detection of biogenic amine-producing bacteria on foods

Biogenic amines are low molecular weight organic bases that can be detected in raw and processed foods. Several toxicological problems resulting from the ingestion of food containing biogenic amines have been described. Biogenic amines are mainly produced by the decarboxylation of certain amino acids by microbial action. Since the ability of microorganisms to decarboxylate amino acid is highly variable, being in most cases strain-specific, the detection of bacteria possessing amino acid decarboxylase activity is important to estimate the risk of biogenic amine food content and to prevent biogenic amine accumulation in food products. Molecular methods for the early and rapid detection of these producer bacteria are becoming an alternative to traditional culture methods. PCR methods offer the advantages of speed, sensitivity, simplicity and specific detection of amino acid decarboxylase genes. Moreover, these molecular methods detect potential biogenic amine risk formation in food before the amine is produced. The aim of the present review is to give a complete overview of the molecular methods proposed in the literature for the detection of biogenic amine-producing bacteria. These genetic procedures allow the introduction of early control measures to avoid the development of these bacteria.     

Detection of biogenic amine producer bacteria in a typical Italian goat cheese

The aim of this study was to research decarboxylating bacterial strains and biogenic amine content in a typical Italian goat cheese (Robiola di Roccaverano). The study was performed on fresh and ripened samples of goat cheese manufactured from industrial and artisanal producers. Sixty-seven bacterial strains isolated showed decarboxylating activity, and Enterococcus faecalis was the most widespread decarboxylating species in all artisanal and industrial products. Pediococcus acidilactici and Enterococcus malodoratus were also identified as biogenic amine producers in Robiola di Roccaverano cheese. All the E. faecalis strains isolated in this study were able to decarboxylate tyrosine. Tyramine was the most abundant biogenic amine in cheese samples, while histamine was the most widespread. High amounts of these two biogenic amines were found in ripened samples (up to 2,067 mg/kg for tyramine and 1,786 mg/kg for histamine), whereas 2-phenylethylamine and tryptamine were present in almost all ripened cheeses at low concentrations. The detection of strains producing biogenic amines and the high concentrations of tyramine and histamine found in ripened Robiola di Roccaverano could represent a potential risk to the consumer.     

Quantitative detection and identification of tyramine-producing enterococci and lactobacilli in cheese by multiplex qPCR

Tyramine is the most abundant biogenic amine in fermented dairy products, in which it is produced through the microbial enzymatic decarboxylation of tyrosine. This activity has been detected in a variety of lactic acid bacteria mainly belonging to the genera Enterococcus and Lactobacillus. This paper describes a culture-independent qPCR method, based on the specific amplification of the tdc gene, for the detection, quantification and identification of bacteria with the ability to produce tyramine. This method was found to be specific and to show a wide dynamic range, thus allowing the quantification of these tdc+ bacterial groups among the complex microbiota of cheese. tdc qPCR was used to follow the development of tdc+ microbiota during the manufacture of a blue-veined cheese (Cabrales) made from raw milk. In this type of cheese, tdc+ enterococci seem to be responsible for the high concentrations of tyramine detected. The method was also used to identify and quantify tdc+ enterococci and lactobacilli in 18 commercially available cheeses. Different types and numbers of these microorganisms were found. Their relationships with the concentration of tyramine and technological factors are discussed.     

Reduction of biogenic amine formation using a negative amino acid-decarboxylase starter culture for fermentation of Fuet sausages

The ability of Lactobacillus sakei CTC494, a negative amino acid-decarboxylase starter culture, to reduce biogenic amine accumulation during sausage fermentation and storage at 4 and 19 degrees C was studied. The effect on the amine formation of the tyramine producer Lactobacillus curvatus CTC371, as a positive strain, was also examined in comparison to a spontaneous fermentation process without starter culture (control batch). The polyamines spermine, spermidine, and diaminopropane were not influenced by the ripening, and their levels slightly decreased in all the batches throughout the storage. Tyramine, cadaverine, and putrescine were the main amines formed during the ripening. The addition of starter culture resulted in a decrease on the biogenic amine formation, depending on the strain inoculated. A great reduction in tyramine content was achieved when L. sakei CTC494 was inoculated, whereas L. curvatus CTC371 only attenuated tyramine accumulation compared with the control batch. Both starters were able to significantly limit the production of putrescine and cadaverine, and they inhibited tryptamine and phenylethylamine formation by the wild microbial flora. Tyramine levels of the control sausages rose during the storage at both temperatures, whereas those of cadaverine only increased at 19 degrees C. On the contrary, sausages manufactured through the starter controlled fermentation did not show changes of amine contents during the storage. The addition of a proper selected starter culture is advisable to produce safer sausages with low contents of biogenic amines.     

Volatile and biogenic amines, microbiological counts, and bacterial amino acid decarboxylase activity throughout the salt-ripening process of anchovies (Engraulis encrasicholus)

Chemical and microbiological parameters were studied during the industrial production of salt-ripened anchovies (Engraulis encrasicholus). Gradual acidification and increases in the proteolysis index and in total volatile basic nitrogen were observed. At the end of the maturing process, the values reached pH 5.55 +/- 0.03, 21.33 +/- 5.82%, and 44.06 +/- 12.47 mg/ 100 g, respectively. In the three studied anchovy batches, the biogenic amines tyramine, histamine, putrescine, cadaverine, and agmatine increased during ripening. The highest values were found in the batch where initial microbial load was highest (batch 1), especially for enterobacteria and enterococci. Tyramine was the most abundant amine, reaching values from nondetectable to 90 mg/kg, whereas histamine did not surpass 20 mg/kg. Among the microorganisms isolated, Enterobacter cloacae, Aerococcus viridans, Kocuria varians, and Staphylococcus chromogenes were able to decarboxylate amino acids and produce biogenic amines in vitro. Most (70.59%) of the microorganisms identified were able to produce histamine, 23.53% were able to produce the diamines putrescine and cadaverine, and only 11.76% were able to produce tyramine, although this substance was the major biogenic amine found in anchovy samples.     

猪肉自然发酵香肠中细菌和乳酸菌对生物胺积累的作用

在猪肉香肠的自然发酵时，检测了选择的污染菌对形成的生物胺的性质和产量的作用。本文主要研究的是在肉腐败过程中胺的变化和微生物生长的变化，而且重点研究的是加入产酪胺的短乳杆菌（L．brevis）和产尸胺的阴沟肠杆菌（E．cloacae）的猪肉发酵香肠在发酵时生物胺的积累。在腐败和香肠发酵时相关微生物和胺的量较多。尸胺的高度积累和大量的不理想肠细菌有关，在香肠发酵时，酪胺的产生并不完全是乳酸菌的作用。另一方面，也涉及到必要的生产香肠的自然发酵菌，污染菌对于腐胺和其它生物胺的作用，现在还不清楚。     

Production of biogenic amines by lactic acid bacteria and enterobacteria isolated from fresh pork sausages packaged in different atmospheres and kept under refrigeration

The occurrence of in vitro amino acid activity in bacterial strains associated with fresh pork sausages packaged in different atmospheres and kept in refrigeration was studied. The presence of biogenic amines in decarboxylase broth was confirmed by ion-exchange chromatography and by the presence of the corresponding decarboxylase genes by PCR. From the 93 lactic acid bacteria and 100 enterobacteria strains analysed, the decarboxylase medium underestimates the number of biogenic amine-producer strains. 28% of the lactic acid bacteria produced tyramine and presented the tdc gene. All the tyramine-producer strains were molecularly identified as Carnobacterium divergens. Differences on the relative abundance of C. divergens were observed among the different packaging atmospheres assayed. After 28 days of storage, the presence of argon seems to inhibit C. divergens growth, while packing under vacuum seems to favour it. Among enterobacteria, putrescine was the amine more frequently produced (87%), followed by cadaverine (85%); agmatine and tyramine were only produced by 13 and 1%, respectively, of the strains analysed. Packing under vacuum or in an atmosphere containing nitrogen seems to inhibit the growth of enterobacteria which produce simultaneously putrescine, cadaverine, and agmatine. Contrarily, over-wrapping or packing in an atmosphere containing argon seems to favour the growth of agmatine producer-enterobacteria. The production of putrescine and cadaverine was associated with the presence of the corresponding amino acid decarboxylase genes. The biogenic amine-producer strains were included in a wide range of enterobacterial species, including Kluyvera intermedia, Enterobacter aerogenes, Yersinia kristensenii, Serratia grimesii, Serratia ficaria, Yersinia rodhei, Providencia vermicola and Obesumbacterium proteus.     

Biogenic Amine Content of Tarhana: A Traditional Fermented Food

Tarhana is a traditional fermented cereal food consumed in nearly all regions of Turkey as a soup having a delicious and desired taste. There are some other products similar to tarhana in Syria, Jordan, Egypt, Iraq, Hungary, Finland, Greece, and Scotland. Fifteen homemade and 5 commercially produced tarhana samples were analyzed for the first time to determine biogenic amine contents. Tyramine was the prevailing biogenic amine. Average tyramine concentrations of homemade and commercially produced tarhana samples were 92.8 and 55.0 mg/kg of tarhana, respectively. Concentrations of biogenic amines were below the permissible limits except for two homemade samples. The pH values of tarhana samples were in the range from 3.43 to 5.03; acidities were from 0.60 to 3.89 g/100 g tarhana (as lactic acid); total dry matters were from 86.42 to 92.32 g/100 g tarhana; and total free amino acid contents were from 0.035 to 1.427 g/100 g tarhana (as leucine).     

植物源生物活性成分控制肉及肉制品中酪胺含量的研究进展

肉及肉制品中的酪胺是在产酪胺菌（粪肠球菌、屎肠球菌等）作用下、适宜条件（25 ℃、pH＜7）下产生的一种生物胺。食用酪胺浓度超过阈值的肉制品会引发高血压、偏头痛等疾病,危害人类健康,因此,抑制肉和肉制品中酪胺形成和降低其含量的方法已成为新的研究热点。抑制食品中酪胺形成的方法有物理法、化学法和生物法,其中利用植物活性成分具有的抑菌/抗氧化性可有效地控制酪胺形成,从而降低酪胺在肉制品中的积累。本文介绍了肉及肉制品中酪胺的形成途径、植物活性成分的特性（抑菌性和抗氧化性）及其结构,以及植物提取物控制酪胺形成、抑制其积累的效果。重点综述了植物源活性成分抑制肉制品中酪胺形成的机理,为降低肉制品中生物胺含量、提高肉及肉制品安全性、促进植物源生物活性成分在食品中的应用提供技术支持。